In nature, stem cells are undifferentiated cells, which are able to differentiate into various functional, mature cells ranging from neuronal cells to muscle cells. Embryonic stem (hereinafter “ES”) cells are derived from the embryo and are pluripotent in nature. ES cells are able to differentiate into a particular cell, tissue or even an organ type depending on the types of stimulus they are subject to.
Development of mouse ES cells was reported in 1981 (Evans et al., Nature 292:151-156; Martin, Proc. Natl. Acad. Sci. U.S.A. 78:7634-7638) and was associated with work on mouse teratocarcinomas. Teratocarcinoma research developed widely in the 1970's and association with the background developmental capacity of the EC (embryonal carcinoma) stem cells led to work on EC cells as models for studies on mammalian cell, tissue and organ differentiation. However, EC cells are neoplastic and they necessarily contain chromosomal aberrations which make their ability to differentiate into various tissue types limited.
Teratocarcinomas can be induced ectopically with blastocyst grafts, and it was hypothesized that pluripotential cell lines could be developed directly from blastocysts instead of tumors. This was the reported development of mouse ES cells in 1981 separately by Martin and Evans et al. The results were stable diploid cell lines that were said to be able to generate every adult tissue type. Teratocarcinomas have also been reported to have been developed from primordial germ cells in mice and from ectopic transplantation of primordial germ cells. In 1992, Matsui et al. published a report on obtaining EG (embryonic germ) cells from mouse primordial germ cells (Cell 70:841-847). EG cells have a developmental capacity very similar to ES cells. As murine ES cells can differentiate into any humoral cell type, mouse ES cells can be used in vitro to study mechanisms which control differentiation of specific cells or tissues. Study of mouse ES cells provides understanding on general differentiation of mammalian cells and tissues, but differences in primate and mouse development and specific lineages limited the use of mouse ES cells as a model for human development.
Pluripotential cell lines have also been derived from testicular carcinomas (Andrews et al., 1984, Lab. Invest. 50:147-162), who reported the derivation of cloned cell lines from human teratocarcinoma which could differentiate in vitro into neurons and other cell types. Cell lines which could differentiate into tissues representative of all three embryonic germ layers were also developed (Pera et al., 1988, Differentiation 39:139-149). These studies on human cellular development showed that these derivations were aneuploid, of limited capacity for spontaneous differentiation into somatic tissue, and different from mouse ES or EC cell phenotype.
Development of primate ES cells from rhesus monkey and marmoset blastocysts have been published (Thomson et al., 1995, Proc. Natl. Acad. Sci. U.S.A. 92:7844-7844; 1996, Biol. Reprod. 55:254-259). Such primate cell lines were diploid, but they closely resembled human EC cells otherwise. These studies on monkey cells showed that work on primates including human EC cells are associated with pluripotent stem cells which are different in phenotype from mouse ES cells, and could be derived from a human blastocyst.
Short term culture and maintenance of cells from human embryos fertilized in vitro was reported by Bongso et al. in 1994 (Hum. Reprod. 9:2110-2117). The isolated cells had morphology expected of pluripotent stem cells, but these early studies did not employ feeder cell support, and it was impossible to achieve long term maintenance of the cultures. Thomson et al. (1998, Science 282:1145-1147) derived ES cells from human blastocyst by removing the trophectoderm by immunosurgery, then plating the inner cell mass onto a mouse embryonic fibroblast feeder cell layer, and following a brief period of attachment and expansion, the resulting outgrowth was disaggregated and replated onto another feeder cell layer. The phenotype of the resulting cells was similar to the human EC cells reported by Pera et al., supra.
Thomson et al.'s studies on primate ES cells showed no evidence that these cells had the capacity for somatic differentiation in vitro. The only evidence for in vitro differentiation was for limited expression of markers characteristic of trophoblast and endoderm formation such as human chorionic gonadotropin alphafetoprotein production. It is difficult to substantiate that cells which produce alphafetoprotein are equivalent to those which make up extraembryonic or embryonic endoderm.
A method for establishing ES cells by culturing undifferentiated primordial germ cells from miscarried fetus, which were 5 to 9 weeks old, has been described (Shamblott et al., 1998, Proc. Natl. Acad. Sci. U.S.A. 95:13726). However, the method did not provide either high quality ES cells or a method for differentiating ES cells derived from long-term stored, frozen embryos to specific cells.
Reubinoff et al., published an article regarding ES cells derived from relatively fresh blastocyst stage embryos, which were 6 days old after fertilization (2000, Nature 18:399-404). Reubinoff reports the propagation of undifferentiated human ES cells and production of human ES cells capable of yielding somatic differentiated cells. However, this method did not provide an efficient method for generating high quality ES cells derived from long-term stored, frozen embryos.
Human EC cells will form teratocarcinomas with derivatives of multiple embryonic lineages in tumors in nude mice. However, the range of differentiation of these human EC cells is limited compared to the range of differentiation obtained with mouse ES cells, and all EC cell lines derived are aneuploid (Andrews et al., 1987, supra). ES cells, on the other hand, are thought to retain greater developmental potential because they are derived from normal embryonic cells in vitro, without the selective pressures of the teratocarcinoma environment. True ES cells should: (i) be capable of indefinite proliferation in vitro in an undifferentiated state; (ii) maintain a normal karyotype through prolonged culture; and (iii) maintain the potential to differentiate to derivatives of all three embryonic germ layers (endoderm, mesoderm, and ectoderm) even after prolonged culture.
Any method that would allow production of human ES cells is desirable, since human ES cell lines would permit easier study of early human development, and the use of such human ES cell lines would enable the development of cell cultures for transplantation, manufacture of bio-pharmaceutical products, and development of biological-based sensors. Importantly, the ability to produce large quantities of human cells has important working applications for the production of substances, such as insulin or factor VIII which currently must be obtained from non-human sources or donors; implantation to treat diseases, such as Parkinson's diseases; tissue for grafting; and screens for drugs and toxins.
It is an objective of the present invention to overcome or at least alleviate some of the problems of the prior art and to provide a more effective and practical method for producing human ES cells, and the following disclosure provides a practical system which meets the needs in the art as described above and provides additional advantages as well.